The goal of this research is to elucidate the mechanisms by which heterotrimeric G proteins modulate the activities of their intracellular effectors. The studies proposed here both build upon and expend the work completed in the first six years of funding of this grant, and break new ground It is proposed that a rate-limiting conformational rearrangement precedes hydrolysis of GTP by Galphai1. To test this hypothesis, the structure of the Galphai1 GTP complex will be determined by time-resolved x-ray diffraction using caged GTP analogs, and the structures of mutant Galpha subunits with abnormally high GTPase activities will be determined. To test whether bond-cleavage is rate-limiting, and to elucidate the structure of the transition state, 18O kinetic isotope effects on the GTPase rate will be measured using labeled substrates, in the presence and absence of the GTPase activating protein (GAP) RGS4. The crystal structure of Galphaq, which has distinctive kinetic properties, shall be determined in complexes that mimic the GTP-bound and transition states. To discover how G protein effectors act synergistically with RGS proteins to accelerate GTP hydrolysis, structures of complexes between GMP phosphodiesterase gamma, the GAP domain of RGS9 and a soluble chimera of Galphai1 and Galphat will be determined. Several experiments are proposed to understand how G proteins activate effectors. Based on the crystal structure of the complex between Galphas and the catalytic core of adenylyl cyclase (AC), a model of allosteric activation was proposed. To test this model, crystallographic and fluorescence spectroscopic studies will be undertaken to determine the structure of AC in its basal, inactive state. To learn how AC is inhibited by Galphai1, crystal structures will be determined of the complex between the N-terminal, C1a module of the AC catalytic domain with Galphai1. Complexes between phosphoinositide-specific phospholipase Cbeta or its constituent domains with Galphaq and Gbetagamma will be crystallized to provide structural insight into the mechanism of signal transduction by Gq. These experiments will elucidate the molecular mechanics that are fundamental to biological processes that control endocrine regulation in both normal and disease states.